The present invention relates generally to methods of treatment of microbial infections of animals, including aquatic animals and fish. The present invention also relates to kits for the application of the method of administering an effective antimicrobial agent to the external surface of an animal.
Aquatic animals are regularly exposed to a medium with a high microbial population. Although the animals have physical and immune system barriers that resist opportunistic infections, such defenses are occasionally breached and an infection results. In the natural environment, the infected animal will overcome the disease and survive, or it will succumb, weaken and die. Since naturally aquatic animals are in limited physical contact with one another, or the water in which they live circulates freely, cross-infection and epidemics are rare unless the water is heavily polluted and/or the animal population is at high density. In the confines of a small aquarium or pond, with ornamental fish or the much larger tanks of fish farming (aquaculture) operations, the possibility of a microbial infection of an entire population is greater. The fish farming, or aquaculture, industry is particularly susceptible to infections of a stock population and the economic consequences of a disease ravaging a tank of fish can be significant. The rapid and effective treatment of a microbial infection, therefore, is essential to save individual fish, and to prevent the spread of disease to healthy animals.
Fish confined to tanks, especially under the high-density conditions prevalent in aquaculture, are sensitive to stress-induced illness. Fish health maintenance for the keeping of ornamental or tropical fish or for the rearing of farmed fish for human consumption, therefore, demands paying close attention to a combination of factors to minimize stress to the fish. The proper environmental conditions of temperature and pH, balanced nutrient delivery and regular provision of food, lack of overcrowding and minimal physical handling contribute to the overall health of fish stock. An imbalance or perturbation of one or more of these factors can significantly increase susceptibility to microbial infection and disease.
Fish microbial infection can be caused by primary or obligate pathogens that are not part of a normal aquatic flora, or by opportunistic pathogens that typically inhabit the water in which fish live. Infectious fish pathogens can establish themselves after any combination of stress to the fish. Physical trauma to the surface or skin of a fish can allow an opportunistic pathogen to penetrate the overlying mucoid and epidermal layers of the skin to reach the underlying tissues. The fish immune system can be compromised by adverse changes in the environmental conditions, such as unacceptable food or excessive handling. Should the water in which the fish are living become heavily contaminated with bacteria, the fish""s own defenses against microbial invasion can be overwhelmed and an infection ensue.
Bacterial and fungal, and to a lesser extent protozoal, diseases are a frequent occurrence in the keeping of ornamental fish. If optimal conditions are not carefully maintained, or an infected fish is brought into the colony, an infectious microorganism can be introduced that will devastate a population of fish. Ornamental fish are susceptible to many of the same bacteria that cause disease in mammals and reptiles. Multiple species from, for example, the bacterial genera Aeromonas, Proteus, Streptococcus, Pseudomonas and Edwardsiella, have been implicated in morbidity and mortality of ornamental fish.
Three methods of treating a microbial infection of fish are typically used. Direct injection of antibiotics into the muscle or blood stream offers a high success rate against systemic infections, but is traumatic for the fish because of the necessary physical handling. Sedation can sometimes help to calm the animal. This procedure, however, is labor intensive and time-consuming, and is only a viable option for the treatment of small quantities of ornamental fish or the breeding stock used in fish-farming. Even then, injection is better reserved for the larger fish species and breeds such as koi, and goldfish, and farmed species such as catfish, salmon, and trout.
The second available treatment is to add antibiotics to fish feed. For the amateur breeder or keeper of ornamental fish, medicated fish foods are available from a veterinarian, but there may be an unacceptable delay in obtaining the medication before a fish succumbs to the infection. There is also evidence of over-use of antibiotics by ornamental fish breeders causing an increase in antibiotic resistance in bacteria. For the fish farmer, medicated foods can raise environmental concerns because of the discharge of untreated farm waste containing antibiotics not consumed by the fish, or which have survived excretion from the fish. The possibility of antibiotic release to the open environment and the potential to select for multiple or specific drug resistant mutants of bacteria requires strict regulation of the use of medicated foods in fish farming. Nitrifying bacteria useful in regulating the ammonia content of a tank can be reduced in number if antibiotics are added in high concentration in a culture tank. Also, the effectiveness of an antibiotic directly added to a fish tank may be limited by divalent ions such as calcium or magnesium that can bind and neutralize some antibiotics, especially tetracyclines and quinolones. Released antibiotics can also alter the ecological distribution of the microbial flora in the vicinity of the water release. A further problem with medicated foods is that sick fish may not ingest the food in the necessary therapeutic amounts or even at all. Even when successfully ingested, this treatment is best reserved for the treatment of internal systemic infections.
Bath treatments, or dips are a practical and simple means of treating surface infections of fish and lesions, such as skin ulcers. Although used to administer anesthetics to fish, or to apply pesticides to treat parasitic arthropod infestations such as sea lice infections of salmon, little use has been made of baths for administering antibiotics directed against microbial infections. Short-term treatment of surface bacterial infections such as fin rot, bacterial gill disease, or columnaris, a disease of the outer skin layer, can be achieved by bathing a fish in a weak potassium permanganate solution. Antibiotic baths, however, are seldom used to treat microbial infections in fish, and then only for surface or superficial infections. Deep ulcers and systemic diseases are typically treated with antibiotics administered by injection, food medication, or by merely isolating an infected fish to prevent the spread of the disease to the remainder of a population.
Unlike lavages that treat only the immediate area of infection, bath or dip treatments immerse the whole animal. The fish may be left in the dip to ensure adequate exposure to the therapeutic agents. However, the bath ideally should be separate from a tank used to rear fish to prevent unnecessary exposure of fish stock to antibiotics. A separate tank also resolves environmental issues about disposal of large volumes of antibiotics from waste tank water.
In addition to fish, aquatic animals kept in tanks are also susceptible to surface bacterial infections. Animals captured or rescued from the wild may have infectious lesions that would benefit from an enhanced antibiotic activity and a simple means to administer an effective but safe therapeutic dose to the external surface of the animal. The limitations inherent in treatment methods of fish, however, apply equally to other aquatic species. Since the aqueous environment prevents the localized topical application of antibiotics unless the animal can be kept dry for a prolonged period on dry land, immersion baths are an important option in the treatment of infections on aqueous animals.
Regardless of the mode of administering an antibiotic solution to an animal, there is a constant possibility of inducing or selecting for antibiotic or multiple drug resistance by the bacteria. The wide-spread over-use of antibiotics in the fish-rearing industry has selected for highly resistant strains of bacteria that no longer respond to conventional antibiotic therapy. Although there is a paucity of data concerning antibiotic resistance in marine organisms, the increasing occurrence of microbial resistance to antibiotics might be expected. Any procedure that would enhance or otherwise supplement the potency of antimicrobial agents, therefore, would be invaluable for the effective treatment of microbial diseases.
The synergistic effects of combining an antibiotic with a chelating agent for the inhibition of bacterial growth have been reported in Wooley et al. Am. J. Vet. Res., 36, 15331535 (1975); Bjorling and Wooley, J. Am. Vet. Ed. Assoc. 181, 596-597 (1982); Wooley et al., Am. Vet. Res. 43, 130-133 (1982); Wooley, R. E. Mod. Vet. Pract. 113-116 (February 1983); Wooley et al., Am. Vet. Res. 44, 2167-2169 (1983); Wooley et al., Am. Vet. Res. 44, 1521-1524 (1983); Wooley et al., Vet. Microbiol. 10, 57-70 (1984); Wooley and Jones, Vet. Microbiol. 8, 271-280 (1983); Wooley et al., Vet. Microbiol. 12, 65-75 (1986); Wooley et al., Am. Vet. Res. 44, 1154-1158 (1983); Wooley et al., Am. Vet. Res. 42, 2010-2013 (1981); Wooley et al. J. Am. Vet. Med. Assoc. 175, 81-818 (1975); Sparks et al, Vet. Res. Comm. 18, 241-249 (1994). Similar compositions have also been disclosed for the flushing and coating of medical devices in U.S. Pat. No. 5,688,516 to Raad et al. incorporated herein by reference in its entirety.
There is a need for methods of treating microbial infections of animals, especially aquatic animals, more especially fish, that allow for reduced doses of antibiotics while maintaining antibiotic efficacy. Antibiotic baths are also required in which fish may be immersed for effective treatment of a microbial infection without toxic or harmful effects to the fish. The methods of treatment of microbial fish infections of the present invention can be adapted for a number of antibiotics biologically active against bacteria and acceptable for use in fish intended for human consumption.
The methods of the present invention are particularly useful for enhancing the potency of an antibiotic when acting against a microbial organism having resistance to the antibiotic.
The present invention, therefore, provides methods of using aqueous antibiotic baths or dips that incorporate compounds to enhance the activity of antibiotics and allow lower effective concentrations of the antibiotic.
The present invention provides novel methods of reducing bacterial infections of aquatic animals, especially of fish maintained in tanks, aquaria, or ponds. The aqueous environment of fish, even when in tanks, would otherwise require large quantities of antibiotics to ensure the fish are bathed in adequate antibiotic concentrations that are effective against the infection, while not toxic to the fish themselves.
The present invention provides methods for reducing a microbial infection of an aquatic animal, comprising the steps of dissolving an antimicrobial composition in a carrier to give an antimicrobial solution, wherein the antimicrobial composition comprises at least one chelating agent and at least one antibiotic, immersing an aquatic animal in the antimicrobial solution, removing an aquatic animal from the antimicrobial solution, and placing an aquatic animal in water not containing the antimicrobial composition.
One embodiment of the present invention, for example, provides a method for reducing a microbial infection of an aquatic animal, comprising the steps of selecting an aquatic animal having a microbial infection, providing an antimicrobial solution comprising the chelating agents EDTA or TRIENE, and at least one antibiotic, adding a pH buffering agent to the antimicrobial solution and adjusting the pH thereof to a value of between about 7.0 and about 9.0, immersing the aquatic animal in the antimicrobial solution and leaving the aquatic animal therein for a period that is effective to reduce the microbial burden of the animal, removing the aquatic animal from the antimicrobial solution and returning the animal to water not containing the antimicrobial solution. The immersion of the aquatic animal in the antimicrobial solution containing the EDTA, antibiotic, or TRIENE and pH buffering agent may be repeated until the microbial burden of the animal is eliminated.
The present invention further provides kits for use in reducing a microbial infection of a marine or freshwater aquatic animal, comprising at least one container having therein at least one chelating agent, and optionally at least one antibiotic, and packaging material, wherein the packaging material comprises instructions directing the use of the kit for administering the antibiotic and chelating agent to inhibit the proliferation of a microbial infection of an aquatic animal.
Additional objects, features, and advantages of the invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying drawing figures, which are briefly described as follows.